Chiral recognition mechanism and enantioseparation conditions of tryptophan enantiomers using ligand exchange chromatography are reported here. The effect of different kinds and concentration of ligands, bivalent copper ion, organic modifier, pH of mobile phase, and temperature on enantioseparation has been evaluated. The results show that the enantioselectivity is strongly affected by the pH and the ligand concentration. Under the optimum condition, baseline separation of the two enantiomers has been obtained on a C₁₈ column with a resolution of 3.42 in less than 30 min using methanol-water solution (20:80 v/v) as mobile phase containing 3.0 mmol•L⁻¹ L-phenylalanine and 0.5 mmol•L⁻¹ copper sulphate. Thermodynamic data (ΔΔH and ΔΔS) obtained by Van’t Hoff plots reveal that the enantioseparation is an enthalpy-controlled process. The mechanism of chiral discrimination is based on the stabilities of the copper(II) binary complexes and their ternary diasteremeric complexes with amino acids formed in solution and stationary phase. The proposed method has been successfully used for the quality evaluation of tryptophan enantiomers.